Synthesis of diverse and useful collections of oligonucleotidies

ABSTRACT

A new technique for generating mixtures of oligonucleotides in a single automated synthesis is taught. The method can be used to prepare mixed oligonucleotides ideally suited for creation of useful mixtures of oligo- or polypeptides or proteins. Additionally, the technique enables insertion and/or substitution and/or deletion of a nucleotide sequence at one or more sites. For protein mutagenesis, a trinucleotide can be inserted or substituted at codon boundaries. The invented technique makes possible the encoding of all possible single amino acid insertions, or any desired mixture of substitutions and insertions.

This application is a continuation of application Ser. No. 08/066,178,filed Sep. 30, 1994, now abandoned, which is a 371 of PCT/US93/03418filed Apr. 13, 1993 and which is a continuation-in part of applicationSer. No. 07/868,489, filed Apr. 15, 1992.

FIELD OF THE INVENTION

The present invention relates to the synthesis of oligonucleotides, andspecifically a new method for generating mixtures of oligonucleotides ina single automated synthesis. The technique enables the generation ofdiverse mixtures of DNA which can be used to prepare large collectionsof oligo- or polypeptides and/or proteins:

BACKGROUND OF THE INVENTION

Methods of preparing DNA of mixed composition are becoming increasinglyimportant in the study of biomolecular function as well as in the searchfor substances with new and useful properties. As DNA synthesistechnology improved in the early 1980's, it became feasible to performmultiple syntheses as a means of generating mixtures ofoligonucleotides. In principle, large and diverse collections could bemade in multiple syntheses. In practice, several investigators realizedthat large numbers of oligonucleotides could be generated in a singlesynthesis by coupling mixtures of mononucleotides, instead of uniquemonomer building blocks. The complexity of the resulting collection or"library" of oligonucleotides is determined by the number of monomerscoupled, and the number of sites at which mixtures of monomers areintroduced.

Oligonucleotides of mixed composition are increasingly being used inprotein mutagenesis for the study of structure and function. Byexpressing DNA sequences of mixed composition, a corresponding libraryof mutant proteins is generated. Allied with appropriate screeningtechniques, such libraries can be searched for substances with alteredproperties, and are therefore useful in the study of biomolecularfunction. The most general class of mutagenesis methods employsoligonucleotides based on the sequence of the wild-type gene, andincorporating modifications that will eventually give rise to anydesired amino acid sequence changes. These methods were recentlyreviewed in the August 1991 issue of Current Opinion in StructuralBiology.

Virtually all genetic studies of protein structure and activity employsubstitution mutations: one or several amino acid side chains arereplaced, but the length of the protein and the spacing of residues areconserved. In order to facilitate the generation of large numbers ofsubstitution mutations in a single experiment, a number of prior arttechniques have been developed (for review, see Botstein, D. & Shortle,D. (1985) Science 229, 1193-1201 and Zoller, M. J. (1991) Curr. Opin.Struct. Biol. 1, 605-610), the most popular of which involve thechemical synthesis of complex mixtures of oligonucleotides which areused either as mutagenic primers for DNA synthesis (see Hermes, J. D.,Parekh, S. M., Blacklow, S. C., Koster, H., & Knowles, J. R. (1989) Gene84, 143-151) or as mutagenic duplex fragments for ligation torestriction fragments (see Matteucci, M. D. & Heynecker, H. L. (1983)Nucl. Acids Res. 11, 3113-3121). To generate the required single aminoacid substitutions, each monomer used for oligonucleotide synthesis is"doped" with small amounts of the three non-wild type mononucleotides.In principle, this method can provide every possible nucleotidesubstitution in a gene segment in a single experiment. Since thedistribution of nucleotide substitutions will follow Poisson statistics,two mononucleotide replacements in the same codon will be relativelyrare at levels of doping that give one or just a few amino acidsubstitutions per mutant gene. Consequently, for practical purposes,this strategy for generating mixtures of mutagenic oligonucleotides canbe expected to yield only one third of all possible amino acidsubstitutions, with the types of amino acid substitutions induced at aparticular position being determined by the sequence of the wild-typecodon. It should also be noted that prior art monomer doping ofoligonucleotides cannot be used to induce other types of changes in DNAsequence, such as insertions or deletions.

A related application of mixed DNA synthesis uses vast collections ofdiverse oligonucleotides in processes directed at discovering substanceswith new and useful properties. Libraries of peptides (Cwirla, S. E.,Peters, E. A., Barrett, R. W., & Dower, W. J. (1990) Proc. Natl. Acad.Sci. USA 87, 6378-6382), RNA (Tsai, D., Kenan, D., & Keene, J. (1992)Proc. Natl. Acad. Sci., USA 89, 8864-8868) and DNA (Bock, L., Griffin,L., Latham, J., Vermaas, E., & Toole, J. (1992) Nature 355, 564-566),all of which were generated from collections of oligonucleotidesprepared by mixed monomer synthesis, have been screened to locatemolecules which bind to particular target substances. In this approach,the utility of peptide libraries is critically dependent on the way inwhich the oligonucleotide mixture is generated. This arises because ofthe degeneracy of the genetic code: amino acids are not represented byequal numbers of trinucleotide codons, some amino acids being encoded byonly one codon, some by as many as six. Therefore, althougholigonucleotides prepared from equal mixtures of all four monomers maycontain each of the 64 trinucleotides, the encoded amino acids arerepresented unevenly, and "stop" codons are unavoidably generated. As aresult, amino acids which are encoded by the largest number of codonsare over-represented at the expense of those encoded by only one or twocodons. By way of example, if a particular type of mutation is desired(for example substitution of only hydrophobic amino acids), theresulting library will contain a high proportion of undesired species.This drawback is particularly critical as the number of positions atwhich substitutions are made increases.

In an attempt to improve the efficiency of synthesizing mixed DNAsequences for preparation of peptide and protein libraries, schemes havebeen introduced in which monomers are mixed in a rational manner. Forexample, Youvan has calculated optimal mixtures of monomers forspecifying particular subsets of amino acids (Arkin, A. P., & Youvan, D.C. (1992) Bio/Technology, 10, 297-300). Use of these mixtures increasesthe proportion of desired amino acids in a peptide or protein library.It does not, however, preclude generating undesired substitutionsarising from particular combinations of monomers. Even with this method,the desired substitutions are usually a fraction of those introduced ateach site. Consequently, as the number of sites altered increases, theproportion of desired mutants in the library decreases.

In recognition of the problems associated with the use of mixtures ofmonomers, Huse has described a method (disclosed in WO 92103461) inwhich DNA synthesis is performed so as to emulate multiple syntheses.This is achieved by carrying out the synthesis on multiple solidsupports which can be mixed and re-divided when necessary. In this way,diverse mixtures of oligonucleotides can be made using monomers, and theproblems associated with the degeneracy of the genetic code avoided. Themethod has two disadvantages: (i) for each synthesis, labour intensivedividing and re-mixing of support material is required, and (ii) thetotal number of different sequences which can be synthesized is limitedby the number of physically separable supports used in the synthesis,which is typically of the order of 108.

In summary, existing methods of synthesis of multiple DNA sequencessuffer several disadvantages:

1. Although every possible nucleotide substitution can be generatedusing oligonucleotides doped with mixed monomers, contiguous two andthree mononucleotide substitutions are extremely uncommon. This isdisadvantageous with regard to protein mutagenesis since each amino acidin a protein is specified by three contiguous nucleotides, and thisstrategy can efficiently generate only approximately one third of allpossible amino acid substitutions for each wild-type amino acid in asingle synthesis.

2. No strategy involving the synthesis of mixtures of oligonucleotides,as taught by the prior art, allows for the generation of mutant proteinswith insertions of one or more codons at more than a single site in thesynthesized oligonucleotide.

3. The degeneracy of the genetic code means that any mixture ofmononucleotides used in mixed DNA synthesis unavoidably givesoligonucleotides containing undesired codons or does not provide alldesired codons. This problem becomes critical as the number of positionsat which mixtures are introduced increases.

4. Methods which simulate multiple syntheses are labour intensive, andthe diversity of sequences which can be generated is limited by thenumber of physically separable supports used.

The present invention provides solutions to these problems and enablesthe preparation of mixed oligonucleotides with a multitude ofapplications in modern molecular biology. For example, mixedoligonucleotides prepared according to the present invention can be usedto generate genes encoding peptide and/or protein libraries.Additionally, trinucleotides are useful in preparing degenerate primersfor the polymerase chain reaction. The invention is particularly usefulfor protein mutagenesis; single-stranded mutagenesis primers anddouble-stranded "cassettes" encoding any combination of amino acids canbe readily prepared by applying the method disclosed herein. The presentinvention also enables substitution, insertion, and deletionmutagenesis.

The method relies on the use of pre-synthesized oligonucleotides andadditionally, specially protected mono- and oligonucleotides, which arecompatible with the most efficient methods of DNA synthesis.Trinucleotide building blocks have been used previously in DNA synthesis(see, for example, Hirose, T., Crea, R., & Itakura, K. (1978) Tet.Lett., 2449-2452; Miyoshi, K., Miyake, T., Hozumi, T., & Itakura, K.(1980) Nucl. Acids Res., 8, 5473-5489) when stepwise coupling yieldswere low and it was more desirable to incorporate the largest possibleoligonucleotide blocks at each step. This earlier work differs from thepresent invention as (i) it relied on inefficient and outdatedphosphodiester chemistry and would therefore not allow multiplecouplings, (ii) it was not directed at generating diverse and usefulcollections of mixed oligonucleotides, and (iii) it did not enableinsertion and deletion mutagenesis.

SUMMARY OF THE INVENTION

The present invention overcomes the limitations in the prior art andprovides a new technique for generating mixtures of oligonucleotides ina single automated synthesis. The method is useful in the systematicmutagenesis of proteins or other important genetic elements. The diverseoligonucleotide collections which can be generated by applying thepresent invention are particularly useful in the preparing peptidelibraries which can be screened for molecules which bind to a particulartarget substance. The present invention can also be used in proteinmutagenesis to encode all possible amino acid substitutions, allpossible single amino acid insertions, all possible amino acid deletionsor any desired mixture of substitutions and insertions.

In its most general form, the present invention allows for the synthesisof DNA molecules using oligonucleotide building blocks. Particularlypreferred is the use of trinucleotides which correspond to amino acidcodons. In the description that follows, trinucleotides are used toillustrate the method, although the use of oligonucleotides of differentlength is not precluded.

Trinucleotides are prepared so as to be compatible with standard methodsof automated DNA synthesis. Most conveniently, the free 5' position isprotected with an acid-labile protecting group (typically4,4'-dimethoxytrityl, DMT), the phosphates are protected as methyl orcyanoethyl esters, the bases are protected as benzoyl (A and C) orisobutyryl (G) amides, and the free 3' position is activated forcoupling as either an O-methyl or O-cyanoethyl N,N-diisopropylaminophosphoramidite. As described below, in some cases it is desirable thatthe 5' position is protected differently. This is readily achievedduring trinucleotide synthesis. The method does not preclude the use oftrinucleotides protected and activated in alternative ways.

The present invention can be used for either stoichiometric orsub-stoichiometric coupling of trinucleotides. In each case, theautomated synthesizer proceeds step-wise to synthesize anoligonucleotide by coupling a sequence of monomers specifying thewild-type DNA sequence. At the desired site, the synthesis programme issuspended, and an altered sequence of steps is effected, as describedbelow.

1. Stoichiometric Coupling

In a first embodiment, one or more trinucleotides are used in place ofmonomers for chemical DNA synthesis. The trinucleotides coupleessentially quantitatively, and can therefore be used for automated DNAsynthesis in the same way as monomer building blocks. In a singlesynthesis, stoichiometric coupling of trinucleotide mixtures providesDNA of any desired complexity with complete control over itscomposition. Stop codons can be avoided, and any combination of aminoacids can be encoded at each position. Replacement of a wild-type codonwith any combination of trinucleotides is readily performed by directingthe DNA synthesizer to access an appropriate mixture at the desired stepin the synthesis. The method is therefore ideal for the generation ofpeptide libraries of defined composition. It is also well-suited topreparing mutant oligopeptides or proteins in which a particular classof amino acids (e.g. hydrophobic) is introduced at one or more sites.

2. Sub-stoichiometric Coupling

By reducing the level of trinucleotide used during DNA synthesis,sub-stoichiometric coupling of suitably protected and activatedtrinucleotides can be used in order to achieve substitution, insertionor deletion mutagenesis. In this format, the present invention is wellsuited to generating mutant proteins bearing single amino acidsubstitutions for the study of structure-function relationships. One ormore trinucleotides are added in an amount that is sub-stoichiometric tothe number of 5' termini on the solid support. If a codon is to beinserted, the 5' end of the added trinucleotide is protected in the sameway as the monomers used in the synthesis. If substitution or deletionis desired, the 5'-end of the trinucleotide bears a specially chosenstable protecting group, hereafter referred to as X. In this context, astable protecting group X is any functionality capable of withstandingthe conditions of automated DNA synthesis, but which can be selectivelycleaved when necessary. The trinucleotide can be added at differentpoints in the sequence of the wild-type gene. The end product is acomplex mixture of oligonucleotides based upon the wild-type sequencebut randomly doped with mixtures of unique or degenerate trinucleotides.Insertion, substitution, or deletion mutagenesis are achieved duringsub-stoichiometric coupling as follows:

(i) Insertion (see FIG. 1)

In a second embodiment to generate insertion mutations, thetrinucleotide is chosen to have one of the commonly used protectinggroups at the 5' position, such as DMT. The small fraction of growingchains that undergo addition of the trinucleotide undersub-stoichiometric coupling conditions are deblocked immediately andthen elongated in all subsequent steps. The net result is the additionof three nucleotides corresponding to a codon having been inserted intoan otherwise wild-type sequence. Synthesis continues. At each site wherean amino acid is to be inserted, another sub-stoichiometric coupling iscarried out with either a unique trinucleotide (to generate one type ofinserted amino acid) or a mixture of trinucleotide phosphoramidites(when up to 19 different residues are to be inserted at a single site).

(ii) Substitution (see FIG. 2)

In a third embodiment to generate substitution mutations duringsub-stoichiometric coupling, the trinucleotide is chosen to have astable 5' protecting group X (as defined above), and a differentialdeprotection scheme is applied. Following trinucleotide incorporation,during the next three monomer additions the 5' protecting group on thetrinucleotide is not removed by the acid treatment that cleaves the5'-DMT group of the coupled monomers. Consequently, the small fractionof growing chains that undergo an addition of the trinucleotide are notelongated. After the addition of three conventionally protected monomersto all other chains, which correspond in sequence to the wild-typecodon, an additional step is carried out to remove the protecting groupX at the end of the trinucleotide. Synthesis continues. At each codonwhere amino acid substitutions are to be generated, another coupling iscarried out with either a unique trinucleotide (to generate one type ofsubstituted amino acid) or a mixture of trinucleotide phosphoramidites(when up to 19 different residues are to be introduced at a singlesite).

(iii) Deletion (see FIG. 3)

In a fourth embodiment, deletions can be made by using a mononucleotidewith a stable 5' protecting group X. The stable 5'-protecting group X(as defined above) delineates one boundary of the deletion and preventssubsequent coupling to the small percentage of chains which acquire it.Subsequent stoichiometric coupling of normal monomers occurs only tothose chains which are deprotected during the course of the synthesis.Removal of the stable protecting group allows subsequent coupling to allchains and defines the second boundary of the deletion. This process canbe repeated many times during one round of oligonucleotide synthesis,producing populations of oligonucleotides with many different deletions.

(iv) Substitution and Insertion

In a fifth embodiment, both substitutions and insertions can be madeduring a single synthesis. In this case, both 5'-X and 5'-DMTtrinucleotides are used during a single oligonucleotide synthesis.Following incorporation of the differently protected trinucleotides, the5'-DMT trinucleotide is deprotected and therefore undergoes subsequentextension, while the 5'-X trinucleotide remains protected and is notelongated. Cleavage of the 5'-X group from those chains which acquiredit allows its subsequent extension. In this way both substitutions andinsertions can be generated in a single synthesis.

The mixed sequences generated using trinucleotides for DNA synthesis canbe used in standard oligonucleotide mutagenesis reactions to produce avery complex mixture of mutant genes. Genetic selection, geneticscreening and nucleotide sequencing of the mutant genes will identifyindividual mutations, and appropriate expression systems will allow forthe production of the corresponding mutant oligo- or polypeptides orproteins.

Trinucleotides (as opposed to oligonucleotides that are not multiples of3 in length) offer the advantage that they are only coupled onto theoligonucleotide at positions that correspond to codon boundaries.Therefore, all sequence changes will be in the correct reading frame.Another advantage is that both substituting and inserting trinucleotidescan be used in the same synthesis, permitting the generation ofextremely complex mixtures of oligonucleotides capable of encoding manymillions of mutant forms of the protein undergoing mutagenesis. Althoughcoupling trinucleotides in the wild-type sequence is useful in proteinmutagenesis, the present method can also be used to coupleoligonucleotides of various lengths. This is an advantage over prior arttechniques where only monomer substitutions were possible.

In its most generalized form, the invention involves coupling modified,activated oligonucleotides to produce sequence degeneracy. The addedoligonucleotide may be of any length, although trinucleotides are ofparticular interest in protein mutagenesis. For generation of insertionmutations, a conventional 5'-DMT protecting group may be used. Forgeneration of substitution or deletion mutations a stable 5' protectinggroup is used with a differential or orthogonal deprotection scheme.

A first object of this invention is to generate a mixture ofoligonucleotides in a single automated synthesis.

A second object of this invention is to generate one or more amino acidsubstitutions in a wild-type sequence.

A third object of this invention is to generate one or more amino acidinsertions in a wild-type sequence.

A fourth object of this invention is to generate one or more amino aciddeletions in a wild-type sequence.

A fifth object of this invention is to generate a mixture of amino acidsubstitutions, deletions and insertions in a wild-type sequence.

A sixth object of this invention is to produce enormous amino acidsequence variations in a wild-type sequence, such as in the vitrorandomization of the variable regions of cloned immunoglobulin genes toproduce more efficient catalytic antibodies.

A seventh object of this invention is to add oligonucleotides (ofvarious lengths) at selected sites in a gene sequence as substitutionsand/or insertions and/or deletions.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic outline of the steps used to synthesize anoligonucleotide mixture containing single codon insertions duringsub-stoichiometric coupling. The trinucleotide is shown by three filledcircles surrounded by a rectangle. Monomeric building blocks used priorto trinucleotide coupling are filled circles. Those added after thetrinucleotide are indicated by hatched circles.

FIG. 2 is a schematic outline of the steps used to synthesize anoligonucleotide mixture containing single codon substitutions duringsub-stoichiometric coupling. The trinucleotide and monomeric buildingblocks are as defined for FIG. 1. X is a particularly stable protectinggroup as defined in the text.

FIG. 3 is a schematic outline of the steps used to synthesize anoligonucleotide mixture containing single codon deletions duringsub-stoichiometric coupling. The grey circle surrounded by a square isthe X-protected mononucleotide building block, where X is a particularlystable protecting group as defined in the text. Filled circles areconventional monomeric building blocks coupled prior to addition of theX-monomer, hatched circles are the three monomeric building blockscoupled following addition of the X-monomer which define a codon. Greycircles are monomeric building blocks coupled following deprotection ofthe X-monomer.

FIGS. 4A and 4B are histograms showing the distribution and frequenciesof alanine and glycine codon-insertion mutations recovered in the genefor staphylococcal nuclease. Two experiments were performed, directinginsertions to different parts of the gene (4A and 4B). The horizontalaxis defines codon boundaries, the vertical axis, the numbers ofmutants. Alanine mutants are shaded, and glycine mutants are the openportion of the bars.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides an efficient method for synthesizingoligonucleotides of mixed sequence, and also for generating insertions,deletions and substitutions in genes of wild-type sequence. Theinvention permits the use of conventional solid-phase synthesizers toproduce a mixture of oligonucleotides in a single automated synthesis.The invention permits the insertion and/or substitution of smallsequences, generally trinucleotides, across a defined segment of acloned gene. When a trinucleotide (or a small oligonucleotide having anucleotide length with a multiple of 3) is used, in-phase codoninsertions or substitutions are achieved in the correct reading frame.

The first embodiment of the present invention is the stoichiometriccoupling of one or more trinucleotides during automated DNA synthesis.Typically, synthesis is carried out using a commercially availableautomated synthesizer. The normal synthesis programme is used until itis necessary to couple the trinucleotide. At this point, the programmeis suspended and the synthesizer is instructed to access a bottle fittedat an additional port containing a prepared solution of thetrinucleotide. The trinucleotide bears protecting groups and anactivated 3' position which are compatible with conventional chemicalsynthesis of DNA. For example, the 5' position of the trinucleotide canbe protected as a DMT ether, and the 3' position activated as aphosphoramidite. Thereafter, synthesis continues in the usual way. Oncompletion of the synthesis, the oligonucleotide is released from thecolumn and the bases and phosphate esters deprotected. If the bases areprotected with the conventional benzoyl and isobutyroyl amides, and thephosphates as b-cyanoethyl esters, treatment with hot, concentratedammonia can be used to bring about complete deprotection. If thephosphates are protected as methyl esters, an additional step must beincluded before the hot ammonia treatment, in which phosphatedeprotection is brought about with, for example, thiophenol, usingwell-established conditions. In a preferred embodiment, mixedoligonucleotides can be prepared by using mixtures of trinucleotidesinstead of a single trinucleotide during the synthesis protocol.Synthesis of oligonucleotides of mixed composition is achieved exactlyas described above, except that a solution containing two or moretrinucleotides is accessed when desired.

The second embodiment of the present invention, shown in FIG. 1, is usedto insert trinucleotides into a wild-type sequence duringsub-stoichiometric coupling. This embodiment is valuable in producingin-phase codon insertions which may be used to generate proteins withmodified structures and functional activities. As shown in FIG. 1,oligonucleotide synthesis is initiated from a nucleotide attached to asolid-phase support and continues from left to right by the coupling ofmononucleotides. When synthesis reaches a position in the wild-typesequence where an insertion is to be made (i.e. at a codon boundary), atrinucleotide corresponding to a specific codon is coupled to a smallpercentage (˜1%) of all growing oligonucleotide chains. The DMTprotecting group on the 5' end of the trinucleotide is then removed, andthree more mononucleotides are added to all of the oligonucleotidechains. At this point, the synthesis has advanced to the next codonboundary in the wild-type sequence, and the cycle of (i)sub-stoichiometric coupling of the trinucleotide followed by (ii)removal of the DMT protecting group is repeated, thereby inserting atrinucleotide at the next target site in the chain. In effect, thewild-type "background" sequence is synthesized with mononucleotidecoupling, whereas all couplings involving a trinucleotide yield aninsertion mutant.

Thus by superimposing sub-stoichiometric couplings of the trinucleotidemixture at positions of codon boundaries on an otherwise conventionalautomated synthesis of a wild-type oligonucleotide of length n, aheterogeneous mixture of oligonucleotides is generated. The exactcomposition of this mixture will depend on the number of trinucleotidecouplings and their coupling efficiency. Whatever its composition,urea-polyacrylamide gel electrophoresis can be used to fractionate themixture on the basis of length, permitting the n+3 band encoding allsingle codon insertions to be separated from the wild-type band andother bands encoding multiple insertions. If desired, oligonucleotidesencoding multiple insertions can also be separated onurea-polyacrylamide gels.

A third embodiment of the present invention is the sub-stoichiometriccoupling of trinucleotides for generation of substitution mutations.Although this embodiment may also work well for the substitution of asmall oligonucleotide of any length, a trinucleotide is discussedbecause of its usefulness in protein mutagenesis. As outlined in FIG. 2,the only modification required is the replacement of the 5'-DMTprotecting group on the trinucleotide with a protecting group 5'-X thatis stable to weak acid and the other conditions used in DNA synthesis,but labile to other mild deprotection conditions. (Various protectinggroups can be used, the following is a partial list: (i) levulinate (seevan Boom, J. H. & Burgers, P. M. J. (1976) Tetrahedron Lett. 4875-4878),(ii) silyl ether (see Ogilvie, K. K., Schifman, A. L., & Penny, C.(1979) Can J. Chem. 57, 2230-2238), (iii) fluoren-9-ylmethoxycarbonyl(Fmoc) (see Xu, Y., Lehmann, C., Slim, G., Christodoulou, C., Tan, Z., &Gait, M. J. (1989) Nucl. Acids Res. Symp. Ser. 21, 39-40), (iv)tert-butyldimethylsilyl, (v) allyloxycarbonyl, (vi)dibromomethylbenzoyl, (vii) 5'-O-b-substituted ethylsulfonyl, (viii)tetrahydropyranyl (Thp), (ix) methoxytetrahydropyranyl (Mthp), (x) 1-(2-chloro-4-methyl)phenyl!4-methoxypiperidin-4-yl (Ctmp), (xi)trityloxyacetyl and (xii) tetraisopropyldisiloxy). After coupling of anX-blocked trinucleotide to 1-3% of chains, the subsequent three monomercouplings add the next wild-type codon to the 97-99% of chains that didnot acquire the trinucleotide, but not to those chains that coupled tothe trinucleotide. At this point in the synthesis, deprotection of allchains (mild-acid to release DMT; dilute aqueous hydrazide if X islevulinate; fluoride if X is a silyl ether; dilute base if X is Fmoc)would yield 1-3% of chains with a substitution of the codon specified bythe trinucleotide and 97-99% of chains that are still wild-type insequence. As with the insertion-generating strategy, repetition of thebasic cycle of 1 sub-stoichiometric trinucleotide coupling followed by 3stoichiometric monomer couplings can be used to introduce mutations ateach position across the gene segment defined by the oligonucleotidesequence. Although purifying mutagenic oligonucleotides away from thosewith the wild-type sequence on the basis of size is not feasible, it ispossible to use trinucleotide blocks containing one or two phosphonateor thiophosphate linkages, permitting purification on the basis ofcharge or chromatographic properties.

A fourth embodiment of the present invention is the use ofdifferentially protected monomers for deletion mutagenesis. As shown inFIG. 3, codon deletions can be generated through the use of X-protectedmononucleotides by sub-stoichiometric couplings followed by fourDMT-monomer addition cycles prior to total deprotection. Again,synthesis proceeds normally until the boundary of a codon which is to bedeleted is reached. At the codon boundary, a sub-stoichiometric amountof a 5'-X-mononucleotide phosphoramidite capable of preserving thewild-type amino acid sequence of the adjacent codon is coupled. Fourcycles of conventional, stoichiometric, mononucleotide coupling to allof the chains which did not receive the differentially protectedmononucleotide serve to add the codon which will eventually be deleted,plus an additional mononucleotide. At this point, the 5'-X protectinggroup is removed and two more rounds of conventional mononucleotidecoupling finish the synthesis of the codon bordering the deletion site.The whole cycle can then be repeated. Eventually, a large population ofoligonucleotides encoding single codon deletions will be produced whichcan then be used to direct mutations as described previously.

These general schemes can be useful in cases where enormous sequencevariation is desired, such as the in vitro randomization of the variableregions of cloned immunoglobulin genes to produce more efficientcatalytic antibodies. Likewise, projects that seek to develop tightbinding ligands via phage display libraries and peptide segment displayon enzymes could make use of the enormous sequence complexity which canbe generated using trinucleotides, especially in the later stages ofoptimization of an initial modestly tight binding sequence. For purposesof simplicity, the substitution embodiment and insertion embodimentdescribed above taught a trinucleotide insertion or substitution. Asdiscussed previously, use of a trinucleotide is of particular interestwhen performing an amino acid substitution or insertion. However, it ispossible to introduce the sequence degeneracy with smalloligonucleotides of any length.

The above disclosure generally describes the present invention. A morecomplete understanding can be obtained by reference to the followingspecific examples which are provided herein for purposes of illustrationonly and are not intended to limit the scope of the invention.

EXAMPLE 1 Synthesis of Trinucleotides¹

A. Synthesis of 5'-DMT-dCBz- PO(OMe)!-dT- PO(OMe)!-dT-3'-P(OMe)(NiPr2)!.

To a solution of dT-3'-Fmoc (930 mg, 2.0 mmol), (prepared from thecorresponding 5'-DMT derivative by trichloroacetic acid-catalyseddetritylation) and 5'-DMT-dT-3'- P(OMe)(NiPr2)! (1.5 g, 2.1 mmol) inanhydrous acetonitrile, was added tetrazole (150 mg, 2.1 mmol). After 30minutes at room temperature, the phosphite was oxidised with t-butylhydroperoxide (0.31 ml of an 80% solution in di-t-butyl hydroperoxide,2.5 mmol), and excess tetrazolophosphoramidite was

¹ Abbreviations: Me=methyl, Bz=benzyl, ^(i) Pr=iso-propyl. For the sakeof convenience, trinucleotides are in some cases abbreviated as, forexample, dTdTdT. quenched with methanol. The solution was evaporated,and the DMT group cleaved by treatment with a solution oftrichloroacetic acid (0.82 g, 5.0 mmol) in dichloromethane. The DMTcation was quenched with 10 mM sodium bicarbonate solution, and the5'-HO-dT- PO(OMe)!-dT-3'-Fmoc was extracted with dichloromethane. Theorganic layer was dried (Na2SO4), filtered, and evaporated, and theresidue purified by silica gel chromatography using a 0-8% gradient ofmethanol in dichloromethane (Rf=0.30 in 10% methanol/dichloromethane),yielding 1.1 g (70%) of 5'-HO-dT-3'- PO(OMe)!-dT-3'-Fmoc.

To a solution of purified 5'-HO-dT-3'- PO(OMe)!-dT-3'-Fmoc (780 mg, 1.0mmol) and 5'-DMT-dCBz-3'- P(OMe)(NiPr2)! (950 mg, 1.2 mmol) in anhydrousacetonitrile, was added tetrazole (83 mg, 1.2 mmol). After 30 minutes atroom temperature, the phosphite was oxidised with t-butyl hydroperoxide(0.19 ml of an 80% solution in di-t-butyl hydroperoxide, 1.5 mmol), andexcess tetrazolophosphoramidite was quenched with methanol. The solutionwas evaporated, and the residue purified by chromatography on basicalumina using a 0-8% gradient of methanol in dichloromethane (Rf=0.42 in10% methanol/dichloromethane), yielding 790 mg (53%) of 5'-DMT-dCBz-PO(OMe)!-dT- PO(OMe)!-dT-3'-Fmoc.

The purified, fully protected trinucleotide (300 mg, 0.2 mmol) indichloromethane was treated with triethylamine (100 mg, 1.0 mmol) atroom temperature for 90-minutes to remove the 3'-Fmoc group. The 3'-OHtrinucleotide (Rf=0.26 in 10% methanol/dichloromethane) was then treatedwith chloro-N,N-diisopropylaminomethoxyphosphine (40 mg, 0.3 mmol) atroom temperature for 30 minutes. Excess chlorophosphine was quenchedwith methanol, the solution was washed with water, dried (MgSO4), andthe trinucleotide phosphoramidite was recovered by precipitation fromhexane, yielding 230 mg (80%) of 5'-DMT-dCBz- PO(OMe)!-dT-PO(OMe)!-dT-3'- P(OMe)(NiPr2)!.

B. Preparation of DNA using 5'-DMT-dCBz- PO(OMe)!-dT PO(OMe)!-dT-3 '-P(OMe)(NiPr2)!.

In all cases, automated DNA synthesis was carried out on either anApplied Biosystems ABI 340B or 380B Synthesizer. The phosphoramidite wasdissolved in anhydrous acetonitrile to a concentration of 10 mM, andfitted to the fifth port of the synthesizer. Following coupling of threemonomers to the column, the trinucleotide was delivered in a doublecoupling procedure,. The coupling yield was determined by measuring therelease of DMT cation. A yield in excess of 95% was obtained. Oncompletion of the synthesis, the heptanucleotide was released from thesolid support and the bases deprotected by treatment with concentratedaqueous ammonia in the usual way. Polyacrylamide gel electrophoresis(20%) and HPLC (C18 column, 0.1M triethylammonium acetate/acetonitrile)confirmed the formation of heptanucleotide and the absence of anyfailure sequences.

EXAMPLE 2 Insertion Mutagenesis

A. General Procedure for Insertion of a Single Trinucleotide into theGene for Staphylococcal Nuclease.

Synthesis used the standard 0.2 mmol synthesis routine, modified toeliminate the capping step after sub-stoichiometric addition of thetrinucleotide. Trinucleotide phosphoramidite (25 mg) was dissolved inanhydrous acetonitrile and the vial attached to the fifth injection portof the synthesizer. Coupling efficiencies of individual monomer andtrinucleotide additions were monitored by the release of the 5'-DMTgroup. The concentration of the unpurified oligonucleotide was estimatedfrom the absorbance at 260 nm.

After synthesis, 10-15 nmol of impure oligonucleotide was phenolextracted, vacuum dried, and re-suspended in 5≠1 of 5 mM NaCl, 1 mMEDTA, 10 mM Tris.HCl, pH 8.1 at 65iC for 30 minutes. An equal volume of95% formamide, 20 mM EDTA, 0.1% bromophenol blue, and 0.1% xylene cyanolwas added, the samples heated at 100iC for 2 minutes, loaded onto a 0.4mm thick by 42 cm long 15-20% polyacrylamide gel, and electrophoresed at750 V until the xylene cyanol was half-way down the gel. The gel wasstained in 2 mg/ml ethidium bromide for 30 minutes, oligonucleotidebands were visualized by UV illumination, and a 0.5-1.0 cm section ofgel immediately above the major band was excised and eluted overnight in300 mM sodium acetate, 5 mM EDTA at 37iC. After brief centrifugation toremove particulates, the oligonucleotide mixture was ethanolprecipitated.

The impure oligonucleotide mixture was radiolabelled with g-32P!ATPusing polynucleotide kinase, in order to confirm the presence of the n+3band and to quantitate its recovery. Approximately 1 pmol of purifiedn+3 oligonucleotide was used to mutagenize a uracil-containing M13derivative phage carrying the gene for staphylococcal nuclease. Mutantplaques were identified using a chromogenic indicator agar, and thenuclease gene of each mutant phage was sequenced in its entirety by thedideoxy method.

B. General Technique for Insertion of One or Two Codons.

A sub-stoichiometric coupling of a mixture of DMT-dGdCdT-phosphoramiditeand DMT-dGdGdT-phosphoramidite was carried out during synthesis ofmutagenic oligonucleotides for the staphylococcal nuclease gene.Following this reaction, which yielded 1-3% coupling, the standardcapping step with acetic anhydride was omitted. (Otherwise, the 97-99%of chains that did not undergo reaction would have been inactivated toadditional couplings.) The subsequent steps of phosphite oxidation anddeprotection of the 5'-DMT group were carried out exactly as inconventional monomer addition cycles. At this point in the synthesis,1-3% of chains had an additional dGdCdT or dGdGdT codon at their 5'ends,whereas the remaining 97-99% were wild-type in sequence. Next, threemonomer addition cycles were carried out so that both the normal lengthchains and the chains with an extra codon received the next wild-typecodon. Again, a codon boundary had been reached; in order to inducesingle codon insertions at this position, another round ofsub-stoichiometric coupling of the trinucleotide mixture was carried outwith omission of the 5' capping reaction. At this point in thesynthesis, 1-3.% of chains had acquired the second insertion of eitherdGdCdT or dGdGdT, 1-3% had acquired the first, less than 0.1% had bothinsertions, and the remaining majority had the sequence of wild-type.Three more monomer addition cycles were then carried out to attach thenext wild-type codon to all chains. Further couplings of thetrinucleotide mixture were carried out after every third monomercoupling until codons had been inserted at all targeted sites. A final6-9 monomer couplings then followed to increase the amount of wild typesequence homology needed for priming second strand synthesis on asingle-stranded DNA template by the oligonucleotide.

C. Use Of dGdCdT Trinucleotide for Single Codon Insertion Mutagenesis.

Insertions of the trinucleotide dGdCdT were made at the codon boundaries64/65, 65/66, and 66/67 of the staphylococcal nuclease gene. When thepurified oligonucleotide mixture was used to mutagenize single-strandedphage, 15% of the resulting phage plaques were deficient in nucleaseactivity. Of the 24 mutant isolates that were sequenced, 11 had a dGdCdTinsertion at 65/66, 11 a dGdCdT insertion at 66/67, one was wild-type,and one had a single nucleotide deletion within the oligonucleotidesequence, presumably due to contaminating n-1 oligonucleotide notremoved by the gel electrophoresis purification step. An identicalexperiment that targeted dGdGdT insertions to these same three sitesgave five dGdGdT insertions at 64/65, ten at 65/66, three at 66/67, onewild-type, one single nucleotide deletion, and four mutations due to theoligonucleotide mis-pairing at other sites in the nuclease gene.

D. Use of dGdCdT and dGdGdT Trinucleotides for Multiple Codon InsertionMutagenesis.

FIGS. 4A and 4B show the results of two experiments in which equimolarmixtures (4.1 mM) of 5'-DMT-dGdCdT-phosphoramidite and5'-DMT-dGdGdT-phosphoramidite were used for insertion mutagenesis. Thehistogram shows the distribution and frequencies of alanine (shown inthe shaded portion of the bar graph) and glycine (shown in the openportion of the bar graph) codon-insertion mutations recovered in thegene for staphylococcal nuclease. In the first experiment, anoligonucleotide of wild-type length n=29 was made, with insertionstargeted to each of the 5 codon boundaries between codons 98 and 103 ofthe staphylococcal nuclease gene. Twenty-four of the 38 mutant plaquescontained single codon insertions, with all sites represented except99/100 (FIG. 4A). Thirteen of the remaining mutants displayed a singlenucleotide deletion within the oligonucleotide sequence consistent withmutagenesis by an oligonucleotide from the contaminating n-1 band. Inaddition, one single nucleotide insertion was found.

In the second experiment, an oligonucleotide of wild-type length n=46.was synthesized to direct insertions to nine of the ten codon boundariesbetween codons 33 and 43 of the staphylococcal nuclease gene. In thiscase 21 of the 37 mutant plaques sequenced contained a single dGdGdT ordGdCdT insertion at a targeted site; the distribution of theseinsertions is shown in FIG. 4B. Again, single nucleotide deletionswithin the oligonucleotide were the major contaminant (twelve isolates),with two single nucleotide insertions plus two larger deletions makingup the remainder.

EXAMPLE 3 Substitution Mutagenesis

A. Synthesis of Trinucleotide Phosphoramidite Coding for Leucine with aFmoc (fluoren-9-ylmethoxycarbonyl) Protecting Group.

A trinucleotide specifying a leucine codon can be custom synthesizedusing standard solution phase chemistry. Re-suspension of the 5'-OHtrinucleotide in dry pyridine followed by incubation with 1.5equivalents of Fmoc-Cl at 0iC for one hour can produce the 5'-Fmocprotected trinucleotide in greater than 50% yield. RP-HPLC can be usedto purify the 5'-Fmoc trinucleotide and its structure can be supportedusing 1H-NMR spectroscopy (Lehmann, C., Xu, Y., Christodoulou, C., Tan,Z. and Gait, M. J. (1989) Nucl. Acids Res. 17, 2379-2389). Standardmethods (see, Balgobin, N. and Chattopadhyaya, J. (1987) Nucleosides andNucleotides 6, 461-463) can be used to phosphitylate the 5'-Fmoctrinucleotide and purify the resulting phosphoramidite. The structure ofthe final product, 5'-Fmoc-dCBz- PO(OMe)!-dT- PO(OMe)!-dT-3'-P(OMe)(NiPr2)! can be supported using 1H and 31P-NMR spectroscopy andcan be confirmed by DNA sequencing of the mutations induced by thetrinucleotide. The lyophilized product should be stored in 25 mgportions under argon in amber vials at -70° C.

B. Codon Substitution with the 5'-O-Fmoc-dCdTdT Phosphoramidite.

Oligonucleotides can be synthesized on a 340B Applied Biosystems DNAsynthesizer using the commercially provided 0.2≠mol synthesis routine.The routine is modified to eliminate the capping step after thesub-stoichiometric addition of the trinucleotide. A step is added inwhich 100 mM DBU (1,8-diazabicyco- 5.4.0!-undec-7-ene) is added from aseparate vial to effect removal of the 5'-Fmoc protecting group of thecoupled trinucleotide after three subsequent mononucleotide couplingcycles. Mononucleotide and trinucleotide coupling efficiencies can bemeasured by monitoring the absorbance of the released DMT and Fmocgroups at 498 and 305 nm respectively. The final oligonucleotide productcontaining a 5'-DMT group can be cleaved from the solid support andpurified away from truncated product by RP-HPLC before standard removalof the remaining protecting groups.

Oligonucleotide concentration can be estimated from the absorbance at260 nm. Approximately 1 pmol of purified oligonucleotide can be used tomutagenize a uracil-containing M13 phage (see Kunkel, T. A. (1985) Proc.Natl. Acad. Sci. USA 82, 488-492) carrying the gene for staphylococcalnuclease. Mutant plaques can be identified by using a chromogenicindicator agar (see Shortle, D. (1983) Gene 22, 181-189), and thenuclease gene of each mutant phage can be sequenced in its entirety bythe dideoxynucleotide chain termination method (see Sanger, F., Nicklen,S. and Coulson, A. R. (1977) Proc. Natl. Acad. Sci. USA 74, 5463-5467).

EXAMPLE 4 Deletion Mutagenesis

A. Synthesis of a Deoxythymidine Mononucleotide Phosphoramidite with a5'-Fmoc (fluoren-9-ylmethoxycarbonyl) Protecting Group.

Suspension of deoxythymidine in dry pyridine followed by incubation with1.5 equivalents of Fmoc-Cl at 0iC for one hour can produce the 5'-Fmocprotected mononucleotide in greater than 50% yield. RP-HPLC can be usedto purify the 5'-Fmoc mononucleotide and its structure can be supportedusing 1H-NMR spectroscopy (Lehmann, C., Xu, Y., Christodoulou, C., Tan,Z. and Gait, M. J. (1989) Nucleic Acids Res. 17, 2379-2389). Standardmethods (see, Balgobin, N. and Chattopadhyaya, J. (1987) Nucleosides andNucleotides 6, 461-463) can be used to phosphitylate the 5'-Fmocmononucleoside and purify the resulting 3'-phosphoramidite. Thestructure of the final product, 5'-FmocO-dT-3'- P(OMe)(NiPr2)! can besupported using 1H and 31P-NMR spectroscopy and can be confirmed by DNAsequencing of the mutations induced using the mononucleotide. Thelyophilized product should be stored in 25 mg portions under argon inamber vials at -70iC.

B. Oligonucleotide Synthesis with Codon Deletion.

Oligonucleotides can be synthesized on a 340B Applied Biosystems DNAsynthesizer using the commercially supplied 0.2 μmol synthesis routine.The routine is modified to eliminate the capping step aftersub-stoichiometric addition of the mononucleotide. A step is included inwhich 100 mM DBU is added from a separate vial to effect removal of the5'-Fmoc protecting group of the coupled trinucleotide after foursubsequent mononucleotide coupling cycles. 5'-Fmoc mononucleotide and5'-DMT-mononucleotide coupling efficiencies can be measured bymonitoring the absorbance of the released DMT and Fmoc groups at 498 and305 nm respectively. The final oligonucleotide product can be cleavedfrom the solid support and purified on the basis of size before standardremoval of the remaining protecting groups.

Oligonucleotide concentration can be estimated from the absorbance at260 nm. Approximately 1 pmol of purified oligonucleotide can be used tomutagenize a uracil-containing M13 phage as above. Mutant plaques can beidentified by using a chromogenic indicator agar, and the nuclease geneof each mutant phage can be sequenced in its entirety by thedideoxynucleotide chain termination method as above.

The invention claimed is:
 1. A method for introducing an insertionmutation at one or more selected positions during the chemical synthesisof a population of DNA molecules of known sequence, said methodcomprising the steps of:(a) sub-stoichiometric coupling ofoligonucleotides to a population of DNA molecules or mononucleotideshaving a sequence consisting of a portion of the known sequence, saidoligonucleotides bearing a 5'-protecting group that is cleaved under aset of conditions used to remove 5'-protecting groups ofmononucleotides, wherein said oligonucleotides further compriseprotected phosphates, protected bases, and a 3'-phosphoramidite group,wherein said coupling is performed without capping, (b) removing all5'-protecting groups under said set of conditions, (c) continuing DNAsynthesis by coupling mononucleotides to said population of DNAmolecules or repeating steps (a) and (b) at a second selected positionand continuing DNA synthesis by coupling of mononucleotides to saidpopulation of DNA molecules to form a population of DNA moleculesconsisting of the known sequence with one or more oligonucleotideinsertion mutations.
 2. The method of claim 1 wherein saidoligonucleotides comprise acid-labile groups protecting free 5'positions, methyl or cyanoethyl ester groups protecting phosphates,benzoyl or isobutyryl amides protecting bases, and 3'-O-methyl orO-cyanoethyl N,N-diisopropylamino phosphoramidite groups.
 3. A methodfor introducing a substitution mutation at one or more selectedpositions during the chemical synthesis of a population of DNA moleculesof known sequence, said method comprising the steps of:(a)sub-stoichiometric coupling of oligonucleotides having a sequence notaccording to the known sequence, said oligonucleotides consisting of anumber of nucleotides, to a population of DNA molecules ormononucleotides, said oligonucleotides bearing first 5'-protectinggroups that are stable to a first set of conditions used to remove5'-protecting groups of mononucleotides, but labile to a second set ofconditions used to remove 5'-protecting groups of mononucleotides,wherein said oligonucleotides further comprise protected phosphates,protected bases, and a 3'-phosphoramidite group, wherein said couplingis performed without capping, (b) sequentially and stoichiometricallyadding said number of nucleotides to those DNA molecules which did notacquire said oligonucleotides bearing first 5'-protecting groups,wherein the nucleotides are added according to the known sequence,wherein the nucleotides bear second 5'-protecting groups, wherein saidsecond 5'-protecting groups are labile to said first set of conditions,and removing said second 5'-protecting groups under said first set ofconditions after each step of adding, (c) removing said first5'-protecting groups on those DNA molecules which acquired saidoligonucleotides using said second set of conditions, and (d)eithercontinuing to synthesize DNA according to said known sequenceusing mononucleotides, or, repeating steps (a) though (c) to generateadditional substitution mutations, and continuing to synthesize DNAaccording to said known sequence using mononucleotides, to form apopulation of DNA molecules consisting of the know sequence with one ormore substitution mutations.
 4. The method of claim 3 wherein saidoligonucleotides comprise acid-labile groups protecting free 5'positions, methyl or cyanoethyl ester groups protecting phosphates,benzoyl or isobutyryl amides protecting bases, and 3'-O-methyl orO-cyanoethyl N,N-diisopropylamino phosphoramidite groups.
 5. The methodof claim 3 wherein said population of DNA molecules of known sequenceencode peptides or proteins.
 6. The method of claim 5 wherein saidnumber is three or a multiple thereof.
 7. The method according to eitherof claims 1 or 3, wherein said oligonucleotides said population of DNAmolecules of known sequence encode peptides or proteins, and saidselected positions are codon boundaries.
 8. The method according toclaim 7 wherein said oligonucleotides bear a 5'-DMT group.
 9. The methodaccording to claim 8 wherein said oligonucleotides comprises amultiplicity of species.
 10. A method for introducing a deletionmutation at one or more selected positions during the chemical synthesisof a population of DNA molecules having a known sequence, said methodcomprising the steps of:(a) sub-stoichiometric coupling of firstmononucleotides to a population of DNA molecules having a sequenceconsisting of a portion of the known sequence of second mononucleotides,said first mononucleotides bearing a first 5'-protecting group that isstable under a first set of conditions used to remove 5'-protectinggroups of mononucleotides, but which is labile to a second set ofconditions used to remove 5'-protecting groups of mononucleotides,wherein said first mononucleotides comprise protected phosphates,protected bases, and a 3'-phosphoramidite group, wherein said couplingis performed without capping, (b) sequentially and stoichiometricallyadding as least third and fourth mononucleotides bearing second5'-protecting groups to those DNA molecules which did not acquire saidfirst mononucleotides bearing said first 5'-protecting group, whereinsaid third and fourth mononucleotides correspond in sequence to theknown sequence, wherein said second 5'-protecting groups are labile to asaid first set of conditions, wherein said third or fourthmononucleotides bearing a second 5'-protecting group bears the samenucleotides moiety as said first mononucleotides, and removing saidsecond 5'protecting groups under said first set of conditions after eachstep of adding, (c) removing the first 5'-protecting group from thoseDNA molecules which acquired the first mononucleotides bearing a first5'-protecting group under said second set of conditions, and (d)eithercontinuing to synthesize DNA according to said known sequenceusing mononucleotides, or, repeating steps (a) through (c) to generateadditional deletion mutations in said DNA molecules of said population,and continuing to synthesis DNA according to said known sequence usingmononucleotides, to form a population of DNA molecules having deletionmutations at one or more selected positions.
 11. The method of claim 10wherein said population of DNA molecules of known sequence encodepeptides or proteins, and wherein said mononucleotides bearing a first5'-protecting group are added at a codon boundary, and wherein saidmononucleotides added in step consist of one or more codons.
 12. Themethod of claim 10, wherein said mononucleotides comprise acid-labilegroups protecting free 5' positions, methyl or cyanoethyl ester groupsprotecting phosphates, benzoyl or isobutyryl amides protecting bases,and 3'-O-methyl or O-cyanoethyl N,N-diisopropylamino phosphoramiditegroups.
 13. The method of claim 10 wherein said first mononucleotidesconform to the known sequence and the fourth mononucleotides have thesame nucleotide moiety as said first mononucleotides.
 14. The method ofclaim 10 wherein said population of DNA molecules of known sequenceencode peptides or proteins.
 15. A method for introducing insertion andsubstitution mutations at one or more selected positions during thechemical synthesis of a population of DNA molecules of known sequence,said method comprising the steps of:(a) coupling of a mixture of firstoligonucleotides and second oligonucleotides consisting of a number ofnucleotides to a population of DNA molecules or mononucleotides having asequence consisting of a portion of the known sequence, said firstoligonucleotides bearing a first 5'-protecting group that is labileunder a first set of conditions used to remove 5'-protecting groups ofmononucleotides, said second oligonucleotides bearing a second5'-protecting group that is stable under said first set of conditions,but labile under a second set of conditions used to remove 5'-protectinggroups of mononucleotides, wherein said first and secondoligonucleotides further comprise protected phosphates, protected bases,and 5'-phosphoramidite groups, (b) removing said first 5'-protectinggroups under said first set of conditions; (c) continuing DNA synthesisby coupling said number of nucleotides according to said known sequenceto said population of DNA molecules which did not acquire said secondoligonucleotides, wherein the nucleotides bear 5'-protecting groups thatare labile under the first set of conditions; (d) removing said second5'-protecting groups under said second set of conditions; (e) continuingDNA synthesis by adding mononucleotides to said population of DNAmolecules according to the known sequence of repeating steps (a) through(d) at a second selected position and continuing DNA synthesis bycoupling of mononucleotides to said population of DNA moleculesaccording to the known sequence to form a population of DNA moleculesconsisting of the known sequence with insertion of substitutionmutations.
 16. A method of preparing a population of DNA molecules ofmixed sequences, which differ from each other at one or more positionswithin the DNA molecules, said method comprising the step of:employing amixed population of trinucleotides, said population comprising aselected combination of trinucleotides, for stoichiometric, essentiallyquantitative incorporation at one or more positions in a population ofDNA molecules of mixed sequences, whereby the population of DNAmolecules formed has a composition defined by the selected combinationof trinucleotides, wherein each of said trinucleotides comprises a3'-phosphoramidite group.
 17. The method according to claim 16 whereinsaid populating of DNA molecules encode peptides or proteins and saidtrinucleotides are incorporated at one or more codon boundaries.